Sea Cucumber Derived Triterpenoid Glycoside Frondoside A: A Potential Anti-Bladder Cancer Drug

Bladder cancer is a highly recurrent disease and a common cause of cancer-related deaths worldwide. Despite recent developments in diagnosis and therapy, the clinical outcome of bladder cancer remains poor; therefore, novel anti-bladder cancer drugs are urgently needed. Natural bioactive substances extracted from marine organisms such as sea cucumbers, scallops, and sea urchins are believed to have anti-cancer activity with high effectiveness and less toxicity. Frondoside A is a triterpenoid glycoside isolated from sea cucumber, Cucumaria frondosa. It has been demonstrated that Frondoside A exhibits anti-proliferative, anti-invasive, anti-angiogenic, anti-cancer, and potent immunomodulatory effects. In addition, CpG oligodeoxynucleotide (CpG-ODN) has also been shown to have potent anti-cancer effects in various tumors models, such as liver cancer, breast cancer, and bladder cancer. However, very few studies have investigated the effectiveness of Frondoside A against bladder cancer alone or in combination with CpG-ODN. In this study, we first investigated the individual effects of both Frondoside A and CpG-ODN and subsequently studied their combined effects on human bladder cancer cell viability, migration, apoptosis, and cell cycle in vitro, and on tumor growth in nude mice using human bladder cancer cell line UM-UC-3. To interrogate possible synergistic effects, combinations of different concentrations of the two drugs were used. Our data showed that Frondoside A decreased the viability of bladder cancer cells UM-UC-3 in a concentration-dependent manner, and its inhibitory effect on cell viability (2.5 μM) was superior to EPI (10 μM). We also showed that Frondoside A inhibited UM-UC-3 cell migration, affected the distribution of cell cycle and induced cell apoptosis in concentration-dependent manners, which effectively increased the sub-G1 (apoptotic) cell fraction. In addition, we also demonstrated that immunomodulator CpG-ODN could synergistically potentiate the inhibitory effects of Frondoside A on the proliferation and migration of human bladder cancer cell line UM-UC-3. In in vivo experiments, Frondoside A (800 μg/kg/day i.p. for 14 days) alone and in combination with CpG-ODN (1 mg/kg/dose i.p.) significantly decreased the growth of UM-UC-3 tumor xenografts, without any significant toxic side-effects; however, the chemotherapeutic agent EPI caused weight loss in nude mice. Taken together, these findings indicated that Frondoside A in combination with CpG-ODN is a promising therapeutic strategy for bladder cancer.


Introduction
Bladder cancer is a commonly diagnosed cancer (3.0% of total cancer cases) and is one of the leading causes of cancer death (2.1% of total cancer deaths). The incidence and mortality rates of bladder cancer have steadily increased, and it has been estimated that there were 573,278 new cases and 212,536 deaths worldwide in 2020 [1]. There are two main types of bladder cancer: muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC), and approximately 75% of bladder cancers are NMIBC [2]. To

Cell Lines and Cell Cultures
The human bladder cancer cell line UM-UC-3 (ATCC ® no. CRL-1749) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The UM-UC-3 cells were maintained in α-MEM medium supplemented with 10% FBS, 1% Nonessential Amino Acid Solution (NAAS), 100 units/mL of penicillin, and 100 µg/mL of streptomycin, in a humidified cell culture incubator at 37 • C with 5% CO 2 .

Cell Viability Assays
Cytotoxicity of single compounds or drug combinations was evaluated using the CCK-8 assay. In brief, UM-UC-3 cells were seeded into the wells of 96-well plates at a density of 5000 cells per well, incubated overnight, and treated with different concentrations of Frondoside A (0.01-2.5 µM) for pre-determined time durations (i.e., 24,48, and 72 h). After the treatments, the UM-UC-3 cells were incubated in the CCK-8 reagent for 3 h and the cell viability was determined according to the manufacturer's instructions. Optical density (OD) was measured at 450 nm using a Bio-Rad microplate reader (Hercules, CA, USA). In the second set of the experiments, cells were also treated for 24, 48, and 72 h with combinations of Frondoside A (0.25-0.75 µM) and CpG-ODN (0.001-1 µM), and the cell viability was assessed using the same procedure as discussed above. In parallel, EPI (10 µM) and PBS (pH 7.4) were also used as positive and blank controls, respectively.

Examination of Synergistic/Antagonistic Effect of Drug Combination
Determination of either synergistic, antagonistic, or additive effects of the compounds when used in combination was performed using the Chou-Talalay method [32,33] based on the date collected in the CCK-8 assays. The combinational index (CI) was calculated with CompuSyn v.1.0 software (ComboSyn, Inc., Paramus, NJ, USA). Synergism is defined as a CI value is less than 0.85, whereas antagonism is defined by a CI value greater than 1.2. A CI value between 0.85 and 1.2 reflects an additive effect.

Cell Migration Assay
The UM-UC-3 cells were cultured to confluence in 6-well plates, and carefully scratched with a plastic pipette tip. Subsequently, the plates were washed twice with PBS (pH 7.4) and incubated at 37 • C in fresh α-MEM containing 1% FBS in pre-determined concentrations of either Frondoside A alone (0.35-1 µM) or Frondoside A (0.5 µM) in combination with CpG-ODN (0.001-1 µM). In parallel, EPI (10 µM) was used as a positive control. The wound-healing area was continuously monitored at different time points (i.e., 12 h and 24 h) using an Olympus inverted microscope (Olympus, Tokyo, Japan).

Cell Cycle Analysis
The cell cycle distribution was analyzed by flow cytometry using PI staining, according to the manufacturer's instructions. More specifically, the UM-UC-3 cells was pre-incubated overnight in 6-well plates (2 × 10 5 cells per well) and were subsequently treated with different concentrations of either Frondoside A alone or Frondoside A (0.5 µM) in combination with CpG-ODN (0.001-1 µM) for 48 h. Subsequently, cells were trypsinized, centrifuged, fixed with 75% ethanol (v/v), washed twice with PBS (pH 7.4), re-suspended in PBS (pH 7.4) containing PI and RNase A at 37 • C for 30 min, and finally quantitatively analyzed using a BD FACS Calibur Flow Cytometer (Franklin Lakes, NJ, USA). Data acquisition was performed using BD Bioscience Cell Quest Pro software (BD Bioscience, Franklin Lakes, NJ, USA). Fractions of cells in G0/G1, S and G2/M phases were determined using a FlowJo software (Version 10.0.7, Tree Star, Inc., Ashland, OR, USA).

Cell Apoptosis and Cell Morphology Analysis
Induction of apoptosis was examined by a FACS-based analysis with an Annexin-V-FITC and propidium iodide (PI) double staining. Specifically, UM-UC-3 cells was seeded in 6-well plates (2 × 10 5 cells per well) and incubated overnight. The different concentrations of either Frondoside A alone (0.35-1 µM) or Frondoside A (0.5 µM) in combination with CpG-ODN (0.001-1 µM) were used to treat the cells for 48 h; in parallel, EPI (10 µM) was used as a positive control. After the treatments, the cells were harvested, re-suspended in 500 µL Annexin V-FITC binding buffer, stained with 5 µL Annexin V-FITC and 10 µL PI in the dark for 20 min, and analyzed using a BD Bioscience FACS Calibur flow cytometer. Induced apoptosis in the UM-UC-3 cells was assessed by Hoechst-33258 staining to observe the morphological changes of the nuclei [34]. The UM-UC-3 cells were seeded in 6-well plates at a density of 8 × 10 4 cells per well overnight and the cells were treated with different concentrations of either Frondoside A alone or the combinations of Frondoside A and CpG-ODN for 48 h, as discussed above. Finally, the cells were stained by Hoechst-33258 for 5 min and observed under a fluorescence microscope (Olympus, Tokyo, Japan).

RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction Analysis
UM-UC-3 cells were seeded in 6-well plates at a density of 4 × 10 5 cells per well overnight. Subsequently, the cells were treated with either Frondoside A (0.5 µM), CpG-ODN (1 µM) individually, or in combination for 48 h; EPI (10 µM) was used as a positive control. After the treatments, total RNA was extracted from the cells using a Trizol Reagent (Life Technologies, Carlsbad, CA, USA). Real-time qPCR (Bio-Rad Laboratories, Hercules, CA, USA) was performed using SYBR Green qPCR master mix (TaKaRa, Dalian, China) according to the manufacturer's protocol. Gene-specific primers used are listed in Table 1. Samples were normalized to the housekeeping gene, GAPDH. The relative RNA levels were determined with the 2 −∆∆Ct method.

In Vivo Bladder Cancer Xenograft Assay
For cancer xenografts, 4-6 weeks old male BALB/c nude mice (nu/nu) were obtained from the Guangdong Medical Laboratory Animal Center and bred in the animal facility. The mice were housed in micro-isolator cages in a filtered-aired laminar flow cabinet and handled under aseptic conditions. UM-UC-3 cells were used to inject into the mice. In brief, 100 µL of the cell suspension (a total of 3 million cells) were injected subcutaneously into the right flank of the mice. When tumors had reached the volume of 70-100 mm 3 , animals were randomly assigned to either a treatment or a control arm (5 mice per group). The animals were treated for 14 days with either Frondoside A (800 µg/kg/day, i.p.), CpG-ODN (1 mg/kg/dose, i.p.), or combination of Frondoside A (800 µg/kg/day, i.p.) and CpG-ODN (1 mg/kg/dose, i.p.) treatment. Control animals were treated with either vehicle alone (0.9% saline) or EPI (1 mg/kg/dose, i.p.). Tumor dimensions and animal weights were monitored every 2 days. Because of animal ethical requirements, animals in all groups were sacrificed when tumor sizes of blank control group were greater than a pre-set endpoint volume of 1000 mm 3 . Once the animals were sacrificed, tumors and the main organs (heart, kidney, liver, spleen, and brain) of the animals were excised and weighed. Finally, the organ coefficient (a common toxicology indicator of ratio of organ and body weight reflecting animal damage) were calculated and the tumor tissues were fixed with 4% paraformaldehyde overnight and H&E stained.

Statistical Analysis
All results were presented as mean ± S.E.M of at least three repeats and plotted using GraphPad Prism software (Version 8.3.0). Statistical analysis was done using ANOVA; differences with p < 0.05 were considered statistically significant.

Frondoside A Inhibits Bladder Cancer Cell Viability and Migration
As shown in Figure 1, Frondoside A reduced cell viability and migration in a concentrationdependent manner ( Figure 1A,B). With the increasing concentrations of Frondoside A (0.01-2.5 µM), the cell viability was significantly decreased (p < 0.05). The IC 50 of Frondoside A was determined to be approximately 0.75 µM for the UM-UC-3 cells (IC 50 = 0.75 µM). When the cells were treated with Frondoside A at concentrations higher than 0.75 µM for 24 h, the inhibition of cell viability was superior to EPI (p < 0.001) ( Figure 1A). In comparison, when the cells were treated with 2.5 µM Frondoside A for 48 h, the degree of inhibition was similar to cells treated with 10 µM EPI for 48 h ( Figure 1A).
It is evident from Figure 1B that with increasing concentrations of Frondoside A (0.35-1 µM), the inhibitory effects on cell migration showed an increasing trend. It should also be noted that at concentration higher than 0.35 µM for both 12 and 24 h treatments, Frondoside A showed more significant inhibitory effects on migration than EPI treatment at a much higher concentration of 10 µM. More specifically, the migration rates of Frondoside A (0.35-1 µM) were 19.31% ± 1.88% to 8.58% ± 1.03% in 12 h, and 43.40% ± 3.13% to 23.77% ± 3.51 in 24 h, while the rates of EPI were 21.15% ± 2.71% and 53.51% ± 3.24% in 12 h and 24 h, respectively ( Figure 1B).

Frondoside A Affects Bladder Cancer Cell Cycle Distribution
As shown in Figure 2, treatment with Frondoside A affected cell cycle distribution in UM-UC-3 bladder cancer cells. Treatments with increasing concentrations (0.35-1 µM) of Frondoside A significantly reduced the fraction of cells in the G0/G1 phase of the cell cycle while an evident increase in the fraction of cells in the S and G2/M phases (see Figure 2A,C) was noted. Additionally, Frondoside A (0.5-1 µM) treatment for 48 h induced a sub-G1 phase arrest, demonstrating that apoptosis is a major contributor to the response seen in this study, a similar effect also seen in cells treated with EPI at 10 µM. Furthermore, UM-UC-3 cells treated with Frondoside A (0.75-1 µM) for 48 h showed increased proportions of cells in sub-G1 phase that was similar or superior to those treated with EPI (10 µM) ( Figure 2B).  Figure 1B).

Frondoside A Affects Bladder Cancer Cell Cycle Distribution
As shown in Figure 2, treatment with Frondoside A affected cell cycle distribution in UM-UC-3 bladder cancer cells. Treatments with increasing concentrations (0.35-1 μM) of Frondoside A significantly reduced the fraction of cells in the G0/G1 phase of the cell cycle while an evident increase in the fraction of cells in the S and G2/M phases (see Figure  2A,C) was noted. Additionally, Frondoside A (0.5-1 μM) treatment for 48 h induced a sub-G1 phase arrest, demonstrating that apoptosis is a major contributor to the response seen in this study, a similar effect also seen in cells treated with EPI at 10 μM. Furthermore, UM-UC-3 cells treated with Frondoside A (0.75-1 μM) for 48 h showed increased proportions of cells in sub-G1 phase that was similar or superior to those treated with EPI (10 μM) ( Figure 2B).

Frondoside A Induces Cell Apoptosis and Change Nuclei Morphology
As shown in Figure 3A, Frondoside A treatment induced cell apoptosis in a concentration-dependent manner while EPI treatment (10 μM) induced cell death through both apoptosis and necrosis. For example, exposure to Frondoside A treatment at 0.5 μM, 0.75 μM, and 1 μM induced 10%, 40%, and 60% of the cells to undergo apoptosis, respectively. Similarly, a concentration-dependent morphological changes were also detected in Fron- . Cell viability was assayed as described in Materials and Methods. Frondoside A significantly suppressed the migration of UM-UC-3 cells, and cell mobility was measured at 0, 12, and 24 h (objective × 4) (B). Data are expressed as means ± S.E.M. * Significantly different at p < 0.05. ** Significantly different at p < 0.01. *** Significantly different at p < 0.001. n.s (not significant). "a" stands for versus blank control; "b" stands for versus EPI (10 µM).

Frondoside A Induces Cell Apoptosis and Change Nuclei Morphology
As shown in Figure 3A, Frondoside A treatment induced cell apoptosis in a concentrationdependent manner while EPI treatment (10 µM) induced cell death through both apoptosis and necrosis. For example, exposure to Frondoside A treatment at 0.5 µM, 0.75 µM, and 1 µM induced 10%, 40%, and 60% of the cells to undergo apoptosis, respectively. Similarly, a concentration-dependent morphological changes were also detected in Frondoside A treated UM-UC-3 cells, showing nuclear fragmentation, chromatin condensation, blebbing of the plasma membrane, and formation of apoptotic bodies ( Figure 3B) under the phase contrast microscope. These morphological changes associated with apoptosis were evidently different from those in EPI treatment at 10 µM ( Figure 3B).

CpG-ODN Enhances the Inhibition of Frondoside A on UM-UC-3 in Cell Viability and Migration Assays, but Has No Significant Altering on Cell Cycle Distribution, Apoptosis and Nuclei Morphology
It can be seen in Figure 4A that, in comparison with individual Frondoside A treatment or CpG-ODN treatment, the combined Frondoside A and CpG-ODN treatment significantly reduced the cell growth at 24, 48, and 72 h. When used in combination, CpG-ODN significantly enhanced the inhibition of cell viability mediated by Frondoside A, indicating that CpG-ODN plays a potentiating role in inhibiting the cell viability. To further investigate the potential synergistic effects of Frondoside A in combination with CpG-ODN, the Chou-Talalay method was used. Based on the Chou-Talalay method, CI values lower than 0.85 or close to 1.2 indicate synergy or additive effects, respectively. Remarkably, Figure 4B shows that Frondoside A was strongly synergistic and additive when used in combination with CpG-ODN. Therefore, the combined concentrations at 0.5 µM of Frondoside A and CpG-ODN (0.001-1 µM) were selected for subsequent experiments to investigate the synergistic effects.
Nutrients 2023, 14, x FOR PEER REVIEW 7 of 20 doside A treated UM-UC-3 cells, showing nuclear fragmentation, chromatin condensation, blebbing of the plasma membrane, and formation of apoptotic bodies ( Figure 3B) under the phase contrast microscope. These morphological changes associated with apoptosis were evidently different from those in EPI treatment at 10 μM ( Figure 3B). The results of cell migration (i.e., cell scratch experiment) showed that the drug combinations of Frondoside A (0.5 µM) and CpG-ODN (0.001-1 µM) also resulted in more pronounced inhibition of cell migration than both EPI (10 µM, positive control) and and caused no detectable increase in the level of apoptosis in comparison with Frondoside A (0.5 µM) treatment alone, the combinations did significantly decrease the G0/G1 phase and increased G2/M phase ( Figure 5A) and induced cell apoptosis and morphological changes in the UM-UC-3 cells ( Figure 5B,C). The combination induced apoptosis rate was estimated to be 20.29% ± 0.81% ( Figure 5B).

CpG-ODN Enhances the Inhibition of Frondoside A on UM-UC-3 in Cell Viability and Migration Assays, but Has No Significant Altering on Cell Cycle Distribution, Apoptosis and Nuclei Morphology
It can be seen in Figure 4A that, in comparison with individual Frondoside A treatment or CpG-ODN treatment, the combined Frondoside A and CpG-ODN treatment significantly reduced the cell growth at 24, 48, and 72 h. When used in combination, CpG-ODN significantly enhanced the inhibition of cell viability mediated by Frondoside A, indicating that CpG-ODN plays a potentiating role in inhibiting the cell viability. To further investigate the potential synergistic effects of Frondoside A in combination with CpG-ODN, the Chou-Talalay method was used. Based on the Chou-Talalay method, CI values lower than 0.85 or close to 1.2 indicate synergy or additive effects, respectively. however, none showed statistical significance. In addition, the level of the cyclin-dependent kinase inhibitor 1A (CDK1A) was unaltered in the UM-UC-3 cells for all treatments with the exception of the EPI treatment at 10 µM ( Figure 6). After EPI treatment (10 µM), the expressions of TP53 and caspase 3 were significantly up-regulated in UM-UC-3 cells, while the Frondoside A (0.5 µM) significantly increased the Bcl-2 expression (Figure 6).

The Suppression of Tumor Growth In Vivo
After treatments with Frondoside A (800 µg/kg/day), CpG-ODN (1 mg/kg/dose), EPI (1 mg/kg/dose) and the combination (Frondoside A and CpG-ODN), the growth of subcutaneously transplanted tumors were inhibited when compared with the blank control (0.9% normal saline). Similar differences were also found in tumor weight at the end of the experiment, both EPI group and the drug combinations of Frondoside A (800 µg/kg/day) and CpG-ODN (1 mg/kg/dose) were more effective than either individual drug alone ( Figure 7A,B). It was evident that the body weight of animals throughout the entire course of the experiments showed a clear downward trend in the EPI group, whereas there was no obvious trend in the other treatment groups ( Figure 7C). In fact, the body weight of mice in the group treated with EPI (1 mg/kg/dose) was significantly reduced (p < 0.05) compared with other groups, suggesting that treatments with Frondoside A and CpG-ODN at the indicated doses did not cause significant side effects in the tested mice ( Figure 7D). It was also noted that there was no significant difference in organ coefficients between the experimental groups and the blank control group (data not shown). Remarkably, Figure 4B shows that Frondoside A was strongly synergistic and additive when used in combination with CpG-ODN. Therefore, the combined concentrations at 0.5 μM of Frondoside A and CpG-ODN (0.001-1 μM) were selected for subsequent experiments to investigate the synergistic effects. The results of cell migration (i.e., cell scratch experiment) showed that the drug combinations of Frondoside A (0.5 μM) and CpG-ODN (0.001-1 μM) also resulted in more pronounced inhibition of cell migration than both EPI (10 μM, positive control) and Frondoside A (0.5 μM) alone ( Figure 4C) at both 12 h and 24 h time points. The effects of the combinations of Frondoside A and CpG-ODN on cell cycle distribution and induce cell apoptosis were also studied. The combinations did not induce significant alteration in the cell cycle and cell apoptosis; as shown in Figure 5, it is clear that while combinations of Frondoside A (0.5 μM) and CpG-ODN (0.001-1 μM) had no significant effects on cell cycle and caused no detectable increase in the level of apoptosis in comparison with Frondoside A (0.5 μM) treatment alone, the combinations did significantly decrease the G0/G1 phase and increased G2/M phase ( Figure 5A) and induced cell apoptosis and morphological changes in the UM-UC-3 cells ( Figure 5B,C). The combination induced apoptosis rate was estimated to be 20.29% ± 0.81% ( Figure 5B).   . Frondoside A significantly suppressed the migration of UM-UC-3 cells, cell mobility was measured at 0, 12 and 24 h (objective × 4) (C), and columns are means; bars are S.E.M. * Significantly different at p < 0.05. *** Significantly different at p < 0.001. n.s (not significant). "a" stands for versus blank control; "b" stands for versus EPI (10 µM); "c" stands for versus Frondoside A (0.5 µM) alone.
As shown in Figure 8, cancer cells in all treatment groups exhibited relatively regular, sparse arrangement and wide cell space in comparison with blank control group (0.9% normal saline) (Figure 8), indicating that all the drug treatments have significant inhibitory effect on tumor growth. Interestingly, a closer look at the cell morphology indicates that the combination treatment group (i.e., Fr A+ CpG-ODN) showed distinctly different cell morphologies from other condition treatment conditions, suggesting that further in-depth investigation into the mechanism for the combination treatment is warranted in future studies (Figure 8).

Frondoside A, CpG-ODN or in Combination Regulates the Expression of Target Genes in TP53 Signaling and Intrinsic Pathway
It is evident from Figure 6 that, in comparison with EPI (10 μM), cells treated with either Frondoside A (0.5 μM), CpG-ODN (1 μM) or in combinations had different effects on the related gene expressions. The qPCR results revealed that the cells treated with Frondoside A (0.5 μM), CpG-ODN (1 μM) or in combinations for 48 h showed elevated levels of Bax and caspase 3 (markers of apoptosis) expressions and higher ratio of Bax/Bcl-2; however, none showed statistical significance. In addition, the level of the cyclin-dependent kinase inhibitor 1A (CDK1A) was unaltered in the UM-UC-3 cells for all treatments with the exception of the EPI treatment at 10 μM ( Figure 6). After EPI treatment (10 μM), the expressions of TP53 and caspase 3 were significantly up-regulated in UM-UC-3 cells, while the Frondoside A (0.5 μM) significantly increased the Bcl-2 expression ( Figure  6).

The Suppression of Tumor Growth In Vivo
After treatments with Frondoside A (800 μg/kg/day), CpG-ODN (1 mg/kg/dose), EPI (1 mg/kg/dose) and the combination (Frondoside A and CpG-ODN), the growth of subcutaneously transplanted tumors were inhibited when compared with the blank control (0.9% normal saline). Similar differences were also found in tumor weight at the end of the experiment, both EPI group and the drug combinations of Frondoside A (800 μg/kg/day) and CpG-ODN (1 mg/kg/dose) were more effective than either individual drug alone ( Figure 7A,B). It was evident that the body weight of animals throughout the entire course of the experiments showed a clear downward trend in the EPI group, whereas there was no obvious trend in the other treatment groups ( Figure 7C). In fact, the body weight of mice in the group treated with EPI (1 mg/kg/dose) was significantly reduced (p < 0.05) compared with other groups, suggesting that treatments with Frondoside A and CpG-ODN at the indicated doses did not cause significant side effects in the tested mice ( Figure 7D). It was also noted that there was no significant difference in organ coefficients between the experimental groups and the blank control group (data not shown).
As shown in Figure 8, cancer cells in all treatment groups exhibited relatively regular, sparse arrangement and wide cell space in comparison with blank control group (0.9% normal saline) (Figure 8), indicating that all the drug treatments have significant inhibitory effect on tumor growth. Interestingly, a closer look at the cell morphology indicates that the combination treatment group (i.e., Fr A+ CpG-ODN) showed distinctly different cell morphologies from other condition treatment conditions, suggesting that further indepth investigation into the mechanism for the combination treatment is warranted in future studies (Figure 8).  Tumor volumes were measured every 2 days to assess the effect of treatments on tumor growth (A). Xenografts were resected, and the weights of the tumors were measured at the end of the observation period (B). The body weight of the mice was monitored every 2 days to observe the drugs toxicity, and the body weight was weighed prior to the death (C,D). Data represent the mean ± S.E.M. * Significantly different at p < 0.05.
Nutrients 2023, 14, x FOR PEER REVIEW 16 o nude mice to establish a xenograft tumor model. Mice were treated with different drugs in 14 da Tumor volumes were measured every 2 days to assess the effect of treatments on tumor growth Xenografts were resected, and the weights of the tumors were measured at the end of the obser tion period (B). The body weight of the mice was monitored every 2 days to observe the dr toxicity, and the body weight was weighed prior to the death (C,D). Data represent the mea S.E.M. * Significantly different at p < 0.05.

Discussion
Bladder cancer is a highly prevalent disease worldwide. Chemotherapy is one of most important treatment options for bladder cancer [3,35], particularly for late-sta bladder cancer with metastasis. Recently, significant progress has been made with n therapeutic options including chemotherapy, targeted therapy, and immunotherapy proved for bladder cancer treatment. However, drug-induced toxicities and drug sistance limit the efficacy and clinical applications of these drugs. Therefore, it is cru to find novel anti-bladder cancer drugs from natural sources, especially marine org isms.
Sea cucumber (Cucumaria frondosa, Holothuroidea), as a highly nutritious medici and edible food, has both strong biological activity and excellent safety [6,8]. In particu sea cucumber triterpenoid glycoside Frondoside A is the most researched group of co pounds due to its potential anti-cancer activities [36]. It has been well established t Frondoside A treatments can specifically target cancer cells, such as pancreatic canc lung cancer, colon cancer, and prostate cancer cell lines while have minimal toxic eff on normal cells [13], suggesting that Frondoside A-a marine organism extract-p sesses a strong anti-cancer activity with less side effects. Hence, in this study, we f investigated the anti-bladder cancer effects of Frondoside A alone or in combination w CpG-ODN on bladder cancer cell viability, apoptosis, cell cycle and migration in vi and subsequently their anti-bladder cancer effects in vivo. In the present study, Fron side A alone or in combination with CpG-ODN decreased the viabilities of UM-UC-3 c in a concentration-dependent manner; When used in combination with both Frondos A (0.75 μM) and concentrations of CpG-ODN (0.001-1 μM), the combination of both dru showed more inhibitory effects on cell growth than EPI (10 μM) at 24, 48, and 72 h. The fore, Frondoside A showed a strong synergistic or additive effect when combined w

Discussion
Bladder cancer is a highly prevalent disease worldwide. Chemotherapy is one of the most important treatment options for bladder cancer [3,35], particularly for late-stage bladder cancer with metastasis. Recently, significant progress has been made with new therapeutic options including chemotherapy, targeted therapy, and immunotherapy approved for bladder cancer treatment. However, drug-induced toxicities and drug resistance limit the efficacy and clinical applications of these drugs. Therefore, it is crucial to find novel anti-bladder cancer drugs from natural sources, especially marine organisms.
Sea cucumber (Cucumaria frondosa, Holothuroidea), as a highly nutritious medicinal and edible food, has both strong biological activity and excellent safety [6,8]. In particular, sea cucumber triterpenoid glycoside Frondoside A is the most researched group of compounds due to its potential anti-cancer activities [36]. It has been well established that Frondoside A treatments can specifically target cancer cells, such as pancreatic cancer, lung cancer, colon cancer, and prostate cancer cell lines while have minimal toxic effect on normal cells [13], suggesting that Frondoside A-a marine organism extract-possesses a strong anti-cancer activity with less side effects. Hence, in this study, we first investigated the antibladder cancer effects of Frondoside A alone or in combination with CpG-ODN on bladder cancer cell viability, apoptosis, cell cycle and migration in vitro, and subsequently their antibladder cancer effects in vivo. In the present study, Frondoside A alone or in combination with CpG-ODN decreased the viabilities of UM-UC-3 cells in a concentration-dependent manner; When used in combination with both Frondoside A (0.75 µM) and concentrations of CpG-ODN (0.001-1 µM), the combination of both drugs showed more inhibitory effects on cell growth than EPI (10 µM) at 24, 48, and 72 h. Therefore, Frondoside A showed a strong synergistic or additive effect when combined with CpG-ODN in UM-UC-3 cell cultures. This effect can be explained, at least partially, by the ability of Frondoside A to play a leading role in inducing cell apoptosis. It was shown that Frondoside A isolated from Cucumaria frondosa enhances the anti-proliferative effect of gemcitabine in pancreatic cancer, demonstrating additive effects when used in combination with gemcitabine [11]. Samir Attoub et al. found that combinations of Frondoside A with oxaliplatin or 5-Fluorouracil were significantly more effective in inhibiting colon cancer cell HT-29 proliferation and triggering apoptosis than using either of the cytotoxic agents alone [37]. Moreover, previous studies have also shown that Frondoside A can potentiate the anti-cancer effect of the conventional therapeutic agents in acute leukemia [21]. Thus, it can be concluded that Frondoside A from sea cucumbers can be used as a promising candidate to enhance the efficacy of anti-cancer drugs.
Apoptosis is one of the key mechanisms of anti-cancer drugs in cancer therapy and has emerged as an effective target for the discovery and development of novel anti-cancer agents [38,39]. Generally, the machinery of apoptosis is complex and involves many signaling pathways, including extrinsic or intrinsic apoptosis pathways. Previous studies showed that the sea cucumber triterpene glycoside Frondoside A induces the apoptosis through regulation of several pro-apoptotic factors, such as caspase-3, -8, and -9, PARP, Bax, p21, and inhibits autophagy in urothelial carcinoma cells [40]. Serget A. Dyshlovoy et al. demonstrated that Frondoside A induces non-classical apoptosis in human Burkitt lymphoma cells in a caspase-independent manner [41]. Although the underlying mechanism of cell apoptosis induction in cancer cells differs from others reported, it is interesting to note that the induction of apoptosis appears to be a common effector response in Frondoside A to cause tumor cell death. We found that Frondoside A induced cell apoptosis and cell cycle arrest in a concentration-dependent manner and showed morphological changes in apoptotic UM-UC-3 cells, such as the characteristic cytoplasmic cell shrinkage, budding of plasma membrane, and chromatin condensation.
Interestingly, we demonstrated that Frondoside A (0.5 µM) induced apoptosis did not alter TP53 and caspase-3 expressions, but up-regulated Bcl-2 gene, whereas when used together with CpG-ODN (1 µM) the drug combination showed elevated levels of Bax, Bcl-2, and ratio of Bax/Bcl-2 (data none showed statistical). In comparison, treatment with EPI (10 µM) could significantly activate the pro-apoptotic functions of TP53 and caspase-3 (p < 0.05). Therefore, our results suggest that Frondoside A and EPI use different apoptosis signaling pathways and different mechanisms in inducing apoptosis in bladder cancer cells. EPI may primarily affect apoptosis-related pathways, while triterpene glycosides Frondoside A affect apoptosis-related or invasion-and metastasis-related pathways. These mechanisms may explain the caspase-independent and TP53-independent character of Frondoside A induced apoptosis in bladder cancer cells. Although several target genes (TP53, Bax, Bcl-2, Caspase 3, and CDK1A) for these induction of apoptosis effects have been identified, which not be potential therapeutic targets of Frondoside A, the precise mechanisms underlying their effects are unknown. We speculate that Frondoside A possibly has other targets that are related to the apoptosis signal pathway. Therefore, the molecular mechanisms of Frondoside A and CpG-ODN may be involved in more complicated cell signaling pathways and molecular networks, which are needed to be further explored to reveal these underlying apoptosis mechanisms in depth.
As tumor metastasis and recurrence is the primary cause of cancer-related mortality among cancer patients, exploring drugs that can inhibit tumor migration is an effective strategy for bladder tumor therapy [42]. In this study, we provided the first evidence that Frondoside A does not only suppress proliferation of the UM-UC-3 cells but also exerts a strong inhibitory effect on the migratory property of the cancer cells. In addition, the anti-metastatic activity of Frondoside A was also significantly enhanced when combined with CpG-ODN. It is possible that Frondoside A can counter cell invasion by inhibiting cell migration and preventing bladder cancer cells becoming metastatic. Although we have demonstrated that Frondoside A has a significant effect on inhibiting bladder cancer cell migration, the specific molecular mechanisms remain to be identified in the future.
To observe the anti-bladder cancer effect of Frondoside A alone or in combination with CpG-ODN on tumor growth in vivo, UM-UC-3 xenografts were grafted in nude mice.
We demonstrated slower tumor growth in all drug treatment groups when compared with the blank control treatment group (0.9% normal saline). Combination of Frondoside A and CpG-ODN treatments resulted in a remarkable potentiation (p < 0.05) of the Frondoside A therapeutic effects, while at the same time minimized the side effects of the treatment. The cancer inhibitory effects of Frondoside A have been observed previously in LNM35 lung, PC-3, DU145 prostate, and MDA-MB-231 breast xenografts in immune-deficient mice. Based on these results, we believe that our data further confirm that Frondoside A in combination with CpG-ODN has the potential to enhance the treatment of bladder cancers.

Conclusions
The sea cucumber triterpene glycoside Frondoside A showed potential anti-bladder cancer activity by inhibiting cell viability and migration, inducing the apoptosis and affecting cell cycle. CpG-ODN significantly improves the efficacy of Frondoside A in antibladder cancer in vitro and in vivo. These interesting results suggest that combined therapy of CpG-ODN with Frondoside A can be a promising approach in treating bladder cancer. This therapeutic strategy should be further considered as a candidate for neoadjuvant, adjuvant, and palliative treatment for bladder cancer patients.
Author Contributions: L.Y. and M.M. conceived and designed the experiments. R.R., G.C. and X.L. performed the experiments. R.R., L.Y. and X.C. analyzed the data. R.R. wrote the paper. L.Y. and X.C. reviewed and edited the paper. All authors contributed to manuscript. All authors have read and agreed to the published version of the manuscript.